Anti-RSV G protein antibodies

ABSTRACT

Individual monoclonal antibodies and fragments that bind a conserved epitope of the G protein of RSV and which are minimally immunogenic when administered to a human subject, are useful in treating RSV infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional applications61/000,469 filed 25 Oct. 2007 and 61/089,401 filed 15 Aug. 2008. Thecontents of these documents are incorporated herein by reference.

TECHNICAL FIELD

The invention is directed to antibodies that are immunoreactive with afunctionally important epitope contained on the G protein fromrespiratory syncytial virus (RSV) that are minimally immunogenic whenadministered to a human subject. These antibodies may be used toincrease resistance of human subjects against RSV infection as well asto diminish the level of infection in individuals already infected or toameliorate the symptoms caused by RSV infection.

BACKGROUND ART

RSV infection has been a longstanding and pernicious problem globally,including the United States, Europe, Australia and Japan. It isparticularly troublesome in premature infants, young children, and theelderly, and indeed for all individuals with a weakened immune system.It is estimated that about two thirds of children below age 1 and almostall children between age 1 and 4 are infected at least once with RSV,with most recovering without any need for medical attention. However,5-10% have prolonged severe infection, a factor believed to bepredisposing to wheezing and asthma-like symptoms later in childhood.RSV has two major surface glycoproteins, F and G. The sole marketedmonoclonal antibody against RSV is only approved for prophylactic use inpremature infants to prevent infection by RSV, and is directed againstthe F protein. This antibody, palivizumab (Synagis®, from MedImmune) isbroadly useful due to conservation of the F protein sequence amongstrains. By contrast, the G protein is quite variable except for acentral “CX3C” domain that is nearly invariant in nearly 100 sequencedstrains. This region includes a motif that has been shown to interactwith the fractalkine receptor. That interaction is believed tocontribute to the prolonged disease course characteristic of RSV bysuppressing an effective immune response to the virus: Tripp, R. A, etal., Nature Immunology (2001) 2:732-738. This region has also been shownto be an antagonist of the Toll-like Receptor 4, which is again believedto contribute to suppressing an effective immune response: Polack, etal., Proc. Natl. Acad. Sci. USA (2005) 102:8996-9001; Shingai, et al.,Int'l Immunology (2008) epub July 8.

Initial attempts at prophylaxis for RSV by vaccination provedcounterproductive. Enhanced disease and pulmonary eosinophilia wereassociated with vaccination with formalin inactivated RSV or with RSV Gglycoprotein and this has been attributed to the above noted conservedsequence in the G protein designated CX3C region which mimics thechemokine fractalkine. (Haynes, L. M., et al., J. Virol. (2003)77:9831-9844.) Passive immunization using antibodies directed to the Gprotein has generally been considered impractical due to the lack ofconservation of the sequence of this protein among strains.

It has subsequently been confirmed by the same group that anti-G proteinantibody responses engendered by RSV infection or vaccination areassociated with inhibition of the binding of the G protein to thefractalkine CX3C receptor and with modulation of RSV G-protein-mediatedleukocyte chemotaxis (Harcourt, J. L., et al., J. I. D. (2004)190:1936-1940) and that inhibition of this binding adversely affects Tcell responses (Harcourt, J. L., et al., J. Immunol. (2006)176:1600-1608). More recent vaccine efforts have avoided the worseningof disease associated with the formalin fixed vaccine, but the immunityconferred by the newer vaccines has been found to wane rapidly (weeks tomonths), consistent with the poor immunological memory to natural RSV:Yu, et al., J. Virol. (2008) 82:2350-2357. Repeated infection is commonfor this virus, unlike many others. The immunosuppressive properties ofthe G protein may be responsible for this effect.

Monoclonal antibodies directed against the G protein have been known forover 20 years. Anderson, L. J., et al., J. Virol. (1988) 62:4232-4238describe the ability of mixtures of F and G protein monoclonalantibodies (mAbs), and of the individual mAbs, to neutralize RSV. ThemAbs relevant to binding G protein, notably 131-2G, were later studiedby Sullender, W., Virol. (1995) 209:70-79 in an antigenic analysis. Thisantibody was found to bind both RSV groups A and B, representing themajor strains of RSV.

In addition, Mekseepralard, C., et al., J. Gen. Virol. (2006)87:1267-1273 summarize earlier papers showing that passivelyadministered antibodies both to F and G protein were protective againstexperimental infection in rodent models. These articles includeRoutledge, et al., J. Gen. Virol. (1988) 69:293-303; Stott, E. J., etal., J. Virol. (1986) 60:607-613; Taylor, G., et al., Immunol. (1994)52:137-142; and Walsh, E. E., et al., Infect. Immun. (1984) 43:756-758.In the instant article, Mekseepralard, et al., noted that a specificmonoclonal antibody raised against the G protein (1C2) requiredglycosylation in order to neutralize the virus in the presence ofcomplement in vitro or when used in vivo in mice. The authors note thatamino acids 173-186 of the G protein are conserved and that 1C2 wasdirected against a conserved region; however, the method for preparingnon-immunogenic antibodies was relatively crude, namely chimerization ofa murine Fab onto a human Fc region.

In addition, Corbeil, S., et al., Vaccine (1996) 14:521-525 demonstratethat the complement system is involved in the protection of mice fromchallenge with RSV after passive immunization with the murine monoclonalantibody 18A2B2, even though this antibody does not show neutralizingcapability in vitro.

PCT publication WO 00/43040 describes the use of anti-Substance Pantibodies in ameliorating the airway inflammation associated withinfection by RSV. The production of Substance P, a known proinflammatorymediator, is enhanced by administration of the G protein of RSV and isabsent in mutants of RSV that are missing the G protein or carry afunction defeating point mutation in the central conserved region:Haynes, et al., J Virol (2003) 77:9831-9844.

U.S. patent publication 2006/0018925 describes and claims antibodies andsmall peptides that are able to block the interaction of CX3C region ofthe G protein with its receptor. These compositions are suggested asuseful for modulating RSV infection and inducing immunity. Althoughhumanization of the murine antibodies employed in the demonstration ofthe therapeutic and prophylactic value of these antibodies is suggested,no such humanized forms were actually produced or described.

PCT publication WO2007/101441, assigned to Symphogen, is directed torecombinant polyclonal antibodies for treatment of RSV infections. Thepolyclonal recombinant antibodies are composed of individual monoclonalantibodies that were isolated from human serum. Table 5 of thispublication describes 12 monoclonal antibodies that are said to bind toa “conserved region” at amino acids 164-176 of the RSV G protein ofsubtype A. Five of these were tested for affinity to the G protein andaffinities in the range of 100-500 pM were found. Two of theseantibodies were tested for neutralizing ability using the plaquereduction neutralization test (PRNT); one showed an EC₅₀ value ofapproximately 2.5 μg/ml and the other failed to display neutralizationcharacteristics at all.

DISCLOSURE OF THE INVENTION

Antibodies that are specifically immunoreactive with the RSV G proteinas compared to the F protein, including those that are immunoreactivewith strains of both groups A and B, that have high affinity for the Gprotein and potent neutralizing ability, have been identified from humandonors confirmed as having been recently infected by RSV. In addition, amurine anti-G protein antibody, originally disclosed by Anderson, etal., J. Virol. (1988) 62:4232-4238, has been modified so as to minimizethe chance of immunological rejection when administered to humansubjects. The antibodies of the invention are useful as therapeuticagents and also to increase resistance to RSV in human subjects.Specifically, antibodies to the conserved motif within positions 160-176of the G protein of subtype A are therapeutically effective in clearingthe virus from subjects that are already infected and in reducing theairway inflammation characteristic of RSV infections, as well as forprophylactic use.

Thus, in one aspect, the invention is directed to monoclonal antibodiesor immunoreactive fragments thereof that bind an epitope withinapproximately positions 160-176 on the G protein of the A strain of RSVand that are minimally immunogenic when administered to a human subject.These antibodies display neutralizing capabilities in standard plaqueforming assays for neutralization of RSV and demonstrate EC₅₀ in suchassays of <500 ng/ml, preferably <200 ng/ml, more preferably <100 ng/ml.The antibodies of the invention also have affinities for the G proteinof RSV-A2 of <1 nM, preferably <500 pM, more preferably <100 pM. Theantibodies of the invention, in one embodiment, bind within 30 residuesof, or directly to, at least a portion of the CX3C chemokine motifcontained in the G protein of RSV, in a region that has a high degree ofamino acid identity across multiple strains of RSV. The CX3C chemokinemotif is at approximately amino acid positions 182-186 of strain RSV-A2and at the corresponding positions of the G protein in other strains. Ithas been found that the relevant region, within which the antibodies ofthe invention bind, is included within residues 160-176 of the G proteinof RSV-A2 and the corresponding positions of the G protein in otherstrains. This region is highly conserved within the A strain andcontains only a few amino acid differences between the A and B strains.A particularly highly conserved region has the sequence HFEVFNFVPCSIC atpositions 164-176 of RSV A2. Preferably, the antibodies of the inventionbind an epitope that includes the sequence FEVFNF or the sequenceVFNFVPCSIC. In one embodiment, the antibodies of the invention areimmunoreactive with this region of conserved amino acid identity and,thus, with G protein of both group A and group B strains of this virus,and therefore with the G-protein of most strains.

For use in the methods of the invention to treat RSV infection or toenhance resistance to RSV, the monoclonal antibodies or fragments of theinvention may be immunoreactive with a multiplicity of strains in bothgroups A and B and a single monoclonal antibody may suffice to have thedesired effect. Alternatively, the subject to be treated or to be maderesistant may be administered more than a single monoclonal antibody, inparticular where one antibody in the protocol is more highly reactivewith the strains of group A and the other more highly reactive with thestrains of group B.

The invention also includes pharmaceutical compositions useful forprophylaxis or treatment including ameliorating inflammation whichcontain as an active agent a single antibody or immunoreactive fragmentof the invention, or no more than two antibodies or fragments of theinvention.

Other aspects of the invention include methods of using the antibodiesto treat RSV in human subjects or to induce resistance in thesesubjects.

The monoclonal antibodies of the invention may be produced recombinantlyand therefore the invention also includes recombinant materials for suchproduction as well as cell lines or immortalized cells and non-humanmulticellular organisms or cells thereof, or microbial cells, for theproduction of these antibodies. In one embodiment, cells obtained fromhuman subjects are produced in “immortalized” form wherein they havebeen modified to permit secretion of the antibodies for a sufficienttime period that they may be characterized and the relevant encodingsequence cloned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the frequency in ppm of antibodies to variousRSV antigens from human subjects. The desired strain-independent anti-Gphenotype (Gab) is quite rare, around 10 parts per million (ppm) overalland as low as 1 ppm in certain subjects. “Mix” refers to antibodiesbinding both F and G; as F and G have no sequence homology, the bindingis likely attributable to shared carbohydrate determinants.

FIG. 2A is a diagram of the RSV G protein indicating the CX3C region andthe location of conserved disulfide bonds. The diagrammatic version isgeneric to all strains, although the specific numbering of positions isslightly different from one strain to the next.

FIG. 2B plots serum binding from RSV exposed subjects against a panel ofoverlapping 12-mer peptides from RSV G protein, revealing poorimmunogenicity of the central conserved region.

FIG. 2C plots polymorphism frequency for a collection of over 75 RSVstrains as a function of position in the G protein, revealing strikingconservation at the central conserved region and at the alternativesplice site that creates a soluble form of the G protein.

FIG. 3 shows the results of probing an illustrative murine monoclonalantibody (131-2G) against an array of peptides with overlappingsequences. This work identifies the epitope to which the mAb binds. Inthe instance illustrated, the epitope is within 30 residues of the CX3Cmotif.

FIGS. 4A-4D: Panels A and B present summary plots of blood from twodonors. Panel A shows a donor that has a useful frequency of Ga/Gbcross-reactive clones. Panel B shows a donor that does not. Each pointin the plot delineates the relative binding to three probes for a singleclone's secreted antibody footprint. Panel C is the quantitative profileof the secreted protein footprint of a single EBV transformed B cell.Panel D shows the profiles of 4 progeny cells from a HEK293 celltransformed with antibody genes from the cell in panel C. This profileis identical to that in panel C, within the precision of the assay asdefined by replicates in panel D.

FIGS. 5A-5B show the sequences of heavy chains (panel A) and lightchains (panel B) for representative antibodies of the invention.

FIGS. 6A-6F show Biacore results on determinations of affinity of twoantibodies of the invention. As shown in panels E and F, antibody 3D3binds the G protein and does not shows a barely detectable off rate.Panels A and D show binding of the antibody to the sensor surface.Panels B and E show the increase in sensor signal as Ga protein flowsacross the surface and is captured by the bound antibody, followed by adecline in signal as the surface is washed with buffer allowing thebound Ga protein to desorb from the surface. Panels C and F similarlyshow on-rates and off-rates for the Gb protein.

FIG. 7 is a graph of the binding of various antibodies of the inventionas compared to the Synagis® F protein-binding antibody as determined inan ELISA assay using live virus to coat the microplate.

FIG. 8 is a graph plotting affinity to G protein on the X-axis againstbinding to virus on the Y-axis. The two abilities are correlated,although 3D3 shows slightly less affinity to live virus than would bepredicted from its affinity to G protein.

FIGS. 9A and 9B show a comparison of binding of several antibodies tostrains A2 and A5.

FIG. 10 shows the results of neutralization assays. The results areshown in terms of number of plaques plotted against μg of antibody.

FIG. 11 shows a comparison of antibody 3G12 of the invention withSynagis® in neutralizing RSV strain B.

FIG. 12 shows a comparison of the prophylactic activity of two inventionantibodies with Synagis® commercial antibody.

FIGS. 13A-13C show therapeutic efficacy of mAb 131-2G in apost-infection murine model of RSV (treatment at day +3 post-infection),including dose dependent reduction in viral load (panel A) along withother measures of reduced lung inflammation: NK cells and PMN cells(panel B) and interferon-gamma (IFNγ) (panel C).

FIG. 14 shows the time course of viral titer in a mouse model treatedwith 3G12, 3D3 or Synagis® antibodies at a low dose that highlights thepotency advantage of the high affinity antibodies of the invention.

FIG. 15 is a dose/response curve measuring the effect of antibodies onRSV copy number in the lungs of RSV-infected mice when treated at day +3after infection.

FIG. 16 shows comparative ability of Synagis®, 3D3 and 3G12 to reduceviral load at the end stages of infection, after treatment at day +3after infection.

FIG. 17 shows the effect of control antibody, anti-F antibody and anti-Gantibody on BAL cells in the lungs of RSV-infected mice. Treatment wasat day +3 post-infection.

FIGS. 18A and 18B show that F(ab′)₂ immunospecific fragments of anti-GmAb are as effective as the intact mAbs in reducing inflammation inRSV-infected mice when given at day +3 post-infection, but are noteffective in reducing viral load.

FIGS. 19A-19C show the effect of anti-G mAbs on the production of IFNγin BAL at various times of administration of the antibody, ranging fromprophylactic (day −1) to day +3 and day +5 post-infection.

FIG. 20 shows antibody titer to the central conserved region of RSV Gprotein from elderly patients infected with RSV. The patients wereselected according to severity of clinical signs and symptoms, severe ormild. The absence of appreciable titer to the central conserved regionis correlated with severe disease.

MODES OF CARRYING OUT THE INVENTION

As used herein, the term “treat” refers to reducing the viral burden ina subject that is already infected with RSV or to ameliorating thesymptoms of the disease in such a subject. Such symptoms includebronchiolitis, airway inflammation, congestion in the lungs, anddifficulty breathing.

The term “confers resistance to” refers to a prophylactic effect whereinviral infection by RSV upon challenge is at least reduced in severity.

“Immortalized cells” refers to cells that can survive significantly morepassages than unmodified primary isolated cells. As used in the contextof the present invention, “immortalized” does not necessarily mean thatthe cells continue to secrete antibodies over very long periods of time,only that they can survive longer than primary cell cultures. The timeover which secretion of antibody occurs need only be sufficient for itsidentification and recovery of the encoding nucleotide sequence.

The phrase “minimally immunogenic when administered to human subjects”means that the response to administration in humans is similar to thatobtained when human or humanized antibodies are administered to suchhumans. It is known that human or humanized antibodies do elicit aresponse in 5-10% of humans treated. This is true even of antibodiesthat are isolated from humans since there is a certain level ofbackground “noise” in an immune response elicited. The immune responsemay be humoral or cellular or both. In particular, elevated levels ofcytokines may be found in this percentage of individuals.

The phrase “conserved region of the RSV G protein” refers to an aminoacid sequence contained within 50 amino acids, preferably 30 aminoacids, more preferably 20 amino acids on either side of the CX3C region,which is illustrated for a particular strain in FIG. 2A. The conservedregion extends mostly at the upstream portion of the G protein from theCX3C-specific region. Thus, using RSV G protein of strain A2 as a model,the conserved region applicable to the antibodies of the inventionextends from approximately residue 160 through 188, preferably 160-176.

The antibodies of the invention have a number of desirable properties.First, they are immunoreactive with G protein from a multiplicity of RSVstrains, and are typically immunoreactive with G proteins both from Atype strains and B type strains. Second, they have quite high affinitiesfor the G protein, some of them in the range of <2 pM. Thus, theantibodies of the invention have affinities of at least 10 nM,preferably 1 nM, more preferably 500 pM, more preferably 100 pM or 50pM, 10 pM or 1 pM and all values between these preferred exemplarypoints. Synagis®, a commercial antibody directed to the F protein, isestablished to have an affinity of about 5 nM. A higher affinityantibody against F protein, Numax™ (motavizumab) is estimated to have anaffinity of about 50 pM. The antibodies of the invention show superiorability to behave as therapeutics, and exhibit the capacity to lower theviral count in lungs at the peak of infection. They also exhibit thisability at a point where typically the infection has run its course.This is particularly useful as subjects recovering from RSV infectionmay continue to shed virus, and thus be able to infect others in apost-clinical setting. The antibodies and fragments thereof also treatthe symptoms of infection, including inflammation in the lungs.

The antibodies of the invention have been obtained in two exemplaryways. In one approach, an existing monoclonal antibody referenced above,131-2G, that is known to be immunoreactive with the conserved region ofthe G protein, was first sequenced and then humanized by fusing a humanconstant region with modified human variable regions (both heavy andlight chains). The variable regions were chosen based on high homologyto the variable regions from the 131-2G antibody, then modified toincorporate the hypervariable amino acids from 131-2G. The methods forsuch humanization are generally known provided the correct selection ofamino acid replacements can be determined. In the case of 131-2G, theoriginal hybridoma line expressed more than one light chain, requiringdetermination of which one was in fact responsible for binding to theRSV conserved motif. This has been determined by the present inventorsand, in one embodiment, the antibodies of the invention are exemplifiedby the humanized form of mAb 131-2G.

In an alternative method, the antibodies of the invention have beenrecovered from RSV exposed human donors using the proprietary CellSpot™method which is described in U.S. Pat. No. 7,413,868, PCT publicationsWO 2005/045396 and WO 2008/008858, all incorporated by reference.

In this method, 40 RSV-infected donor samples were analyzed, in aprocess yielding ˜500,000 antibody-producing cells per blood sample.Thus, in total, there were ˜20,000,000 different B cells analyzed forproduction of antibodies which are specific to the conserved region ofthe G protein. Only ˜10% of the donors had a useful frequency of Ga/Gbspecific clones (i.e., strain independent), and such clones were onlypresent at ˜1/50,000 cells even in the highest frequency specimens.Overall, the frequency of the desired cells was ˜0.003%, which is lowenough to be impractical to recover by standard methods but readilyaccessible using CellSpot™. FIG. 1 shows the spectrum of reactivities toRSV antigens for 24 donors. As shown in this figure, even in thoseindividuals where antibodies crossreacting with both A and Bstrain-derived G protein were found, the prevalence of these antibodiesis much smaller than that of antibodies immunoreactive with F protein orwith Ga or Gb alone. A surprisingly large number of clones recognizedboth the F and G protein (denoted “mix”), which are likely recognizingshared carbohydrate determinants. Affinities of such anti-carbohydrateantibodies are typically poor and were excluded from furtherconsideration. The highest affinity antibody found within this cohort ofdonors, with an affinity of 1 pM, came from one of the donors with avery low frequency of Ga/Gb specific clones, ˜1 ppm. That is, findingthis highly favorable clone would have been unlikely withoutcomprehensive screening of the full repertoire from all donors.

In order to perform this screen, B cells were immortalized withEpstein-Barr Virus and assessed according to the above-described methods(see Example 2 for details). Successful B cells were identified and thenucleotide sequences encoding the identified monoclonal antibodies wereobtained and sequenced. These were then manipulated recombinantly toproduce antibodies in a mammalian cell line.

An important aspect of the G protein function resides in a secreted formof the protein, s(G), created by an alternative splice site near residue50. Engineering virus to lack s(G) resulted in reduced level ofpulmonary infiltrating cells (Maher, et al., Microbes Infect. (2004)6:1049-1055). Conversely, priming mice with s(G) augments IL-5production and lung eosinophilia (Johnson, et al., J Virol (1998)72:2871-2880). Accordingly, suppressing the activity of s(G) isimportant for effective treatment of RSV. Achieving that goal requires ahigh affinity antibody, as is generally known in the art (e.g., U.S.Pat. No. 7,083,950). Since the central conserved region is specificallyimplicated in the function of s(G) as an immuno-modulatory agent, aneffective antibody against s(G) should target this motif.

Our survey of the human B cell repertoire from RSV exposed subjects wasunbiased in its search for antibodies that bind to the G protein fromboth strains A and B (Ga/Gb cross-reactive antibodies). Because thesurvey was comprehensive (40 subjects, ˜500,000 B cells examined fromeach), it is a striking finding that all of the Ga/Gb cross-reactiveantibodies binding linear epitopes suitable for mapping recognizeepitopes within a few residues of each other, within the centralconserved region. This region is known to be poorly immunogenic, assummarized in FIG. 2B (Plotnicky-Gilquin, et al., Virology (2002)303:130-137), consistent with the low frequency of high affinity clonesto this region reported here. We have further characterized this regionby examining the published sequences of G proteins from >75 RSVisolates. Most residues of the protein show several to manypolymorphisms in the collection. Two regions are strikingly free ofpolymorphisms: the alternative splice site that creates s(G) and thecentral conserved region to which all Ga/Gb cross-reactive antibodiesbind (FIG. 2C). That is, we have discovered that a region which ishighly conserved, indicating critical functionality, is also poorlyimmunogenic. A variety of mechanisms may account for that poorimmunogenicity, for example absence of nearby proteolytic cleavage sitessuitable for effectively presenting the region in combination withhistocompatibility antigens for display to the rest of the immunesystem. Whatever the mechanism, this surprising result is clear: thoseviruses that have survived show low immunogenicity to this region. Wetherefore predicted that augmenting the immune system's activity againstthis region, by passive transfer of suitable antibodies, would beefficacious, and this has proven to be the case in our animal models.The alternative splice site, although equally conserved, is notunusually low in immunogenicity suggesting that its importance is onlywith regard to creation of s(G), thus making it a poor target forpassive immunotherapy.

Production of the human or humanized antibody of the invention isaccomplished by conventional recombinant techniques, such as productionin Chinese hamster ovary cells or other eukaryotic cell lines, such asinsect cells. Alternatively, techniques are also known for producingrecombinant materials, including antibodies, in plants and in transgenicanimals, for example in the milk of bovines, or in microbial or plant orinsect derived single cell systems.

In addition, since the nucleotide sequences encoding the antibodies areavailable, the relevant fragments which bind the same epitope, e.g.,Fab, F(ab′)₂ or F_(v) fragments, may be produced recombinantly (or byproteolytic treatment of the protein itself) and the antibody may beproduced in single-chain form. A variety of techniques for manipulationof recombinant antibody production is known in the art.

For use in therapy, the recombinantly produced antibodies or fragmentsare formulated into pharmaceutical compositions using suitableexcipients and administered according to standard protocols. Thepharmaceutical compositions may have as their sole active ingredient amonoclonal antibody or fragment of the invention, especially amonoclonal antibody or fragment that is crossreactive with G protein ofboth A and B strains. Alternatively, two monoclonal antibodies may bethe sole active ingredients wherein one more strongly reacts with the Astrain G protein and the other more strongly with the B strain Gprotein. In all of these cases, additional therapeutic agents may bepresent, including one or more antibodies that is immunoreactive withthe F protein or other therapeutic agents that are effective against RSVor inflammation. Thus, anti-inflammatories such as both steroidal andnon-steroidal anti-inflammatory compounds may be included in thecompositions. Also, the compounds may include nutritional substancessuch as vitamins, or any other beneficial compound other than anantibody.

In one embodiment, when the formulations for administration are used inorder to increase resistance to infection, complete antibodies,including the complement-containing Fc region are employed. Typically,the antibodies are administered as dosage levels of 0.01-20 mg/kg ofhuman subjects or in amounts in the range of 0.01-5 mg/kg orintermediate amounts within these ranges. In one embodiment, amounts inthe range of 0.1-1.0 mg/kg are employed. Repeated administrationseparated by several days or several weeks or several months may bebeneficial. Boosters may also be offered after one or two or five or tenyears.

In another embodiment, for a therapeutic effect in order to reduce viralload, complete antibodies, containing the complement-containing Fcregion are also employed. The amounts administered in such protocols areof the order of 0.001-50 mg/kg or intermediate values in this range suchas 0.01, 1 or 10 mg/kg are employed. Repeated administration may also beused. The therapeutic treatment is administered as soon as possibleafter diagnosis of infection, although administration within a few daysis also within the scope of the invention. Repeated administration mayalso be employed. In order to reduce the inflammatory response in thelungs, only the immunospecific fragments of the antibodies need beemployed. Dosage levels are similar to those for whole antibodies.Administration of mixtures of immunospecific fragments and entireantibodies is also included within the scope of the invention.

Administration of the antibody compositions of the invention istypically by injection, generally intravenous injection. Thus,parenteral administration is preferred. However, any workable mode ofadministration is included.

The formulations are prepared in ways generally known in the art foradministering antibody compositions. Suitable formulations may be foundin standard formularies, such as Remington's Pharmaceutical Sciences,latest edition, Mack Publishing Co., Easton, Pa., incorporated herein byreference. The formulations are typically those suitable for parenteraladministration including isotonic solutions, which include buffers,antioxidants and the like, as well as emulsions that include deliveryvehicles such as liposomes, micelles and nanoparticles.

The desired protocols and formulations are dependent on the judgment ofthe attending practitioner as well as the specific condition of thesubject. Dosage levels will depend on the age, general health andseverity of infection, if appropriate, of the subject.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLE 1 Cloning and Humanization of 131-2G

Cloning and sequencing of mAb 131-2G. Total mRNA was extracted from131-2G hybridoma according to the manufacturer's directions (RNeasy™kit: Qiagen Santa Clarita, Calif.). Seven family-specific 5′ VγFR1primers designed to target the VH1 through VH7 gene families of Igγ, andone consensus 3′ Cγ1 primer were used to amplify and sequence thevariable region of 131-2G heavy chain. One consensus 5′ Vk primer wasdesigned to amplify each of the Vk families, and one reverse primerspecific to the kappa constant region were used to amplify and sequencethe kappa light chain. The VH and VL transcripts were amplified from 100ng total RNA using reverse transcriptase polymerase chain reaction(RT-PCR).

Two PCR reactions were run for the 131-2G hybridoma: one for light chainkappa (κ) and one for gamma heavy chain (γ1). The QIAGEN® OneStep RT-PCRkit was used for amplification, (Qiagen Catalog No. 210212). Theextracted PCR products were directly sequenced using specific constantregion primers. The derived sequences were compared to known germlineDNA sequences of the Ig V- and J-regions using the V-BASE2 and byalignment of VH and VL genes to the mouse germ line database. Sequenceanalysis: from the nucleotide sequence information, data regarding V andJ gene segment of the heavy and light chain of 131-2G were obtained.Based on the sequence data new primer sets specific to the leadersequence of the Ig VH and VK chain of 131-2G were designed. V gene usageand sequence analysis: Heavy chain genes of 13-12G were from the VH1germline gene family, the germline gene for the D region is DSP2.2 andthe J region was from the JH3 germline. Light chain genes were fromVkappa 1_((K1A5)) and Jkappa4, germline gene families.

131-2G uses a V segment of the IgH-VJ558 VH1 family: M  G  W  S   W  I  F   L  F  L   L  S  G  T   A  G  V   H  S  E 1ATGGGATGGA GCTGGATCTT TCTCTTCCTC CTGTCAGGAA CTGCAGGTGT CCACTCTGAG V  Q  L  Q   Q  S  G   P  E  L   V  K  P  G   T  S  V   K  I  S 61GTCCAGCTGC AACAGTCTGG ACCTGAACTG GTGAAGCCTG GAACTTCAGT GAAGATATCC C  K  A  S   G  Y  S   F  T  G   F  T  M  N   W  V  K   Q  S  H 121TGCAAGGCTT CTGGTTATTC ATTCACTGGC TTCACCATGA ACTGGGTGAA GCAGAGCCATG  K  N  L   E  W  F   G  L  I   N  P  F  N   G  N  T   G  Y  N 181GGAAAGAACC TTGAGTGGTT TGGACTTATT AATCCTTTCA ATGGTAATAC TGGCTACAAC Q  K  F  K   G  K  A   T  L  T   V  D  K  S   S  S  T   A  F  M 241CAGAAGTTCA AGGGCAAGGC CACATTAACT GTAGACAAGT CTTCCAGCAC AGCCTTCATG E  L  L  S   L  T  S   E  D  S   A  V  Y  Y   C  A  R   S  G  K 301GAGCTCCTCA GTCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG ATCGGGAAAA S  Y  D  Y   E  A  W   F  T  Y   W  G  Q  G   T  L  V   T  V  S 361TCCTATGATT ACGAGGCCTG GTTTACTTAC TGGGGCCAAG GGACTCTGGT CACTGTCTCT  A 421GCA 131-2G uses a V segment of the IκV1 subgroup: D  I  V  M   T  Q  T   T  L  S   L  P  V  S   L  G  N   Q  A  S 1GATATTGTGA TGACACAAAC TACACTCTCC CTGCCTGTCA GTCTTGGAAA TCAAGCCTCC I  S  C  R   S  S  Q   T  I  V   H  T  N  G   N  T  Y   L  E  W 61ATCTCTTGCA GATCTAGTCA GACCATTGTA CATACTAATG GAAACACCTA TTTAGAATGG Y  L  Q  K   P  G  Q   S  P  K   L  L  I  Y   K  V  S   N  R  F 121TACCTGCAGA AACCAGGCCA GTCTCCAAAG CTCCTGATTT ACAAAGTTTC CAACCGATTT S  G  V  P   D  R  F   S  G  S   G  S  G  T   D  F  I   L  N  I 181TCTGGGGTCC CAGACAGGTT CAGTGGCAGT GGATCAGGGA CAGATTTCAT ACTCAATATC S  R  V  E   A  E  D   L  G  V   Y  Y  C  F   Q  G  S   H  V  P 241AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TATTACTGCT TTCAAGGTTC ACATGTTCCA F  T  F  G   S  G  T   K  L  E   I  K  R 301 TTCACGTTCG GCTCGGGGACAAAGTTGGAA ATAAAACGGA

Humanization of mAb 131-2G. The binding of an Antibody (Ab) to itscognate Antigen (Ag) is a highly specific interaction. This specificityresides in the structural complementarity between the Ab-combining siteand the antigenic determinant. Ab-combining sites are made up ofresidues that are primarily from the hypervariable or complementaritydetermining regions (CDRs); occasionally, residues fromnon-hypervariable (or framework) regions influence the overall domainstructure and, hence, the combining site.

The mouse VH gene segment repertoire is twice the size of that in humansand contains more functional genes, compared with the human IgH locus.The mouse and the human loci bear no large-scale similarity to eachother. The first two CDRs of VH and VL domains have a small repertoirestructure of main chain conformation known as the canonical structures.The existence of a particular canonical structure is mainly determinedby the length of the CDRs and the presence of key residues at particularsites in the sequence. The same canonical structure combinations of VH1family (VH1 1-2) are shared between the members of human VH1 and mouseVH1 families. Based on the sequence analysis of heavy and light chainsof 131-2G and the fact that 131-2G uses V segments of IgH-VJ558 VH1family and Igκ1 family, both chains were aligned and compared to themembers of the human VH1 and VK1 families. Sequence homology was foundto be 70% and 77% identity to the germ line sequence of Human VH1-8 andVk1-18 respectively. These germ lines were picked as the human frameworkfor the humanized 131-2G mAb.

Epitope mapping of mAb 1312G. Western Blot analysis using RSV lysate andpurified Ga protein suggested that 131-2G recognizes a linear epitope.The binding domain of 131-2G was mapped using a set of overlappingpeptides derived from the RSV-GA2 protein sequence. FIG. 2A diagrams theG protein sequence, including location of the conserved CX3C motif. Inorder to obtain a fine epitope mapping, a scan was performed on a familyof 12-mer Ga derived peptides, each shifted by one residue. An array ofsuch peptides was probed with 131-2G mAb, at 1 μg/ml. Binding of 131-2Gwas detected by goat anti mouse peroxidase-labeled antibody incombination with the super signal chemiluminescence detection system(Pierce, Rockford Ill., USA). As summarized in FIG. 3, the 131-2Gantibody reacts with 8 consecutive peptides spanning the RSV-Ga proteinfrom residue 157 to 176. The epitope recognized by 131-2G is within thepeptide sequences (157) SKPNNDFHFEVF (169) and (169) HFEVFNFVPCSI (176).Based on the common sequence from the 8 peptides, the 131-2G bindingdomain was mapped to residues 164-168.

Three methods were used to characterize the affinity of 131-2G andanalogous human mAbs. First, binding signal was measured for a fixedamount of antibody probed against serial dilutions of antigen in anELISA format. The midpoint of this titration curve is an approximationof the affinity. In the case of 131-2G, that midpoint is 4 nM. Second,the affinity of 131-2G was measured by Biacore analysis at a commercialanalytical laboratory; based on the ratio of on-rate to off-rate, theaffinity was calculated as 7 nM. Third, dilution of the Ga protein onCellSpot™ beads with serum albumin reduces the opportunity for multiplecopies of the protein to interact with the antibody footprint. Theresulting suppression of multi-dentate avidity effects from the rawsignal allows rank ordering of a set of clones for affinity, relative toa known standard. This measure of affinity can be used to compare thehuman antibodies to 131-2G and efficiently select for high affinityclones. All of these methods are improved by availability of aconsistent source of G protein antigen. In our early studies, antigenwas extracted from virus infected cells. Due to variability in thequality of antigen prepared this way, we developed a recombinantexpression system for producing the G protein, which proved to be morereliable.

EXAMPLE 2 Isolation of Human B Cells Secreting Antibody to RSV-Ga/Gb

Peripheral blood mononuclear cells from 40 adults with confirmed RSVinfection were surveyed for human B cells producing anti-viralantibodies. Subjects with the desired antibodies against RSV attachmentG protein were used for cloning of anti RSV-G specific mAbs. The resultof the survey was that ˜10% of the subjects had a frequency of thedesired cells greater than 1 in 100,000. Even those with a lowerfrequency, however, were of interest and in fact the highest affinityantibody identified came from a donor with a very low frequency of thedesired B cell type, ˜1 ppm.

To accomplish the survey and recovery of rare favorable cells, we usedthe previously described CellSpot™ technology. The CellSpot™ assaymethod effectively shrinks an ELISA equivalent assay down to a virtualwell of near single cell dimensions by capturing secreted IgG from asingle cell as a footprint in the vicinity of the cell. As a result,millions of cells can be readily analyzed. Further, by use ofmicroscopic multiplexing reagents (combinatorially colored fluorescentlatex microspheres, cf U.S. Pat. No. 6,642,062), each clone's secretedantibody footprint can be characterized in detail for specificity and/oraffinity using multiple biochemical probes. The fidelity of thequantitative assay is sufficient to enable rescue of extremely rarefavorable cells from the survey population, with the cloned expressioncell showing a phenotype consistent with the original identifying assay.

The screening criteria were: binding to G protein from both of the twomajor strain families, denoted Ga and Gb, and not binding to the Fprotein (the other major viral coat protein). Affinity rank ordering ofclones can also be accomplished by diluting the antigen on the bead withserum albumin. This reduces the chances for multi-dentate binding to thesecreted IgG footprint (an “avidity” effect), thus selecting for higherintrinsic affinity. G protein was purified from Vero cells infected withone or the other of the two RSV strains.

Applied to human B cells, the method begins by depleting non-B cellsfrom PBMCs using standard magnetic separation methods. Cells wereresuspended in IMDM/20% HI-FCS at 1e6/ml; EBV (direct pelleted from thesupernatant of infected B95-8 cells) was added at 1:100 dilution, andthe cells incubated 2 hr at 37° C. Excess virus was washed away, andcells either: cultured at 2e6/ml in IMDM, 20% HI-FCS, 20% Giant celltumor conditioned medium, 2 μg/ml CpG (ODN2006), and 10 ng/ml IL-10 forsurveying only, or further selected for surface IgG using magneticpositive selection. Cells were cultured at 200-300 cells/well onirradiated human lung cells (MRC-5, 5,000 cells/well) in IMDM, 20%HI-FCS, 20% Giant cell tumor conditioned medium, 2 μg/ml CpG (ODN2006),and 10 ng/ml IL-10. Medium was supplemented every 2-3 days. One half ofthe contents of the wells were assayed in CellSpot™ at day 6. Theremaining cells in the small number of wells positive by the surveyassay were then diluted to 10, 5, 1, and 0.5 cells/well with the samefeeder cells and culture conditions. After 4-5 days these limitingdilution plates were again assayed by ELISA or CellSpot™.

Contents of positive wells at limiting dilution were then processedusing Reverse Transcriptase-PCR to recover the encoding polynucleotidefor the antibody heavy and light chains. Total time from thawing PBMCsto recovery of the encoding mRNA sequence via RT-PCR was 10-12 days.

FIG. 4 shows illustrative data from this experiment. Examples ofCellSpot™ profiling of favorable and unfavorable donor blood samples isillustrated in panels A and B. The profile of a favorable clone atinitial detection is shown in panel C, along with replicate profiles inpanel D of antibody secreted from progeny of a HEK293 cell transformedwith cDNA cloned antibody derived from that cell. The profiles areidentical within the precision of the assay, indicating successfulrecovery of the favorable clone.

As was shown in FIG. 1, the majority of anti-RSV antibodies are directedto the F protein or to an antigenic determinant shared by F and G (mostlikely carbohydrate since the two proteins have no sequence homology).Of the G specific antibodies, most bind only Ga or only Gb, consistentwith the known high sequence variability of the G protein. Overall, ˜20million individual B cells were surveyed. The 12 most promisingantibodies were recovered by RT-PCR. Overall, then, the frequency offavorable clones is below 1 in 1 million, and over 50 million ELISAequivalent assays were needed to find those rare clones. The CellSpot™technology thus enabled a more comprehensive survey of clones than wouldotherwise be practical. The quality of the resulting clones is superiorto those found by more limiting screening, and the consensus features ofthat high quality set reveal unanticipated features of the desiredantibodies.

EXAMPLE 3 Cloning of Human Antibodies to RSV-Ga/Gb

Amplification of rearranged Ig Heavy and Ig Light genes from positiveELISA wells was accomplished using semi-nested polymerase chain reaction(PCR). For amplification of a priori unknown V-gene rearrangements, acollection of family-specific V-gene primers was constructed, whichrecognize nearly all V-gene segments in the human Ig Locus. The 5′primers were used together with primer mixes specific for the Cγ, Cκ andCλ gene segments. The clonality of the limiting dilution RSV-G specificB cells was unequivocally determined by sequence comparison of V-geneamplificates from distinct progeny cells, and the amplified full lengthV-gene rearrangements were cloned into IgG expression vectors. Thismethod was also useful to address additional issues, such as V-, D-, andJ-gene usage and the presence and pattern of somatic mutations.

Methods. Total mRNA from the isolated human B cells was extracted usinga commercially available RNA purification kit (RNeasy™; Qiagen(Germany)). Reverse transcription-PCR was done by using total RNApreparations and oligonucleotides as primers. Three PCR reactions wererun for each sample: one for light chain kappa (κ) one for light chainlambda (λ), and one for gamma heavy chain (γ). The QIAGEN® OneStepRT-PCR kit was used for amplification, (Qiagen Catalog No. 210212). Inthe coupled RT-PCR reactions, cDNA is synthesized with unique blend ofRT enzymes (Omniscript™ and Sensiscript™) using antisense sequencespecific primer corresponded to C-κ, C-λ or to a consensus of the CHIregions of Cγ genes, RT is preformed at 50° C. for 1 hour followed byPCR amplification of the cDNA by HotStarTaq DNA Polymerase for highspecificity and sensitivity. Each PCR reaction used a mixture of 5′sense primers. Primer sequences were based on leader sequences of VH, VKand VL. PCR reactions were run at 95° C. for 15 minutes, initial hotstart followed by 20 cycles of 95° C. for 30 seconds (denaturation), 60°C. for 45 seconds (annealing) and 72° C. for 1 minute (elongation).

Nested PCR for detection and cloning of the variable Ig fragments intoexpression vectors. In the second round, an aliquot of 5 μl of the firstamplification reaction was applied. The primers used carry the 5′BglIIand 3′ XbaI restriction sites. Thirty PCR cycles were performed.Identical conditions were used for the first and second rounds ofamplification. Five microliters of each reaction were loaded andseparated on a 1% agarose gel and then stained with ethidium bromide.The V-C PCR product is predicted to amplify rearranged fragments of VHand VL, 500 and 450 bp respectively. PCR bands with a molecular size ofapproximately 500 bp indicated a positive result. PCR products werepurified (Qiagen gel purification kit catalog number 28704) and theextracted PCR products were directly sequenced using specific constantregion primers. The sequences of the cloned fragments were confirmed bysequencing plasmids prepared for recombinant production.

FIG. 5A shows the amino acid sequences of the heavy chains of theantibodies of the invention isolated from human subjects as well as ofhumanized 131-2G, including variable region, the D and J joiningregions, the framework (FR) and complementarity determining (CDR)regions. All of the listed antibodies are immunoreactive with the Gprotein from both the A and B strains except for antibody 3F9, which isimmunoreactive only with G protein from strain A. FIG. 5B shows similarsequence information for the light chains of these antibodies. Dashes inthe sequence listings represent alignment corrections in the genesequences of different lengths.

The PCR fragments described above were digested and cloned intoindividual expression vectors carrying the constant region of humangamma 1, or of human kappa or lambda, for in vitro antibody productionin mammalian cells. The expression vectors coding for heavy and lightchains were co-transfected into the 293 (human kidney) cell line(Invitrogen). The expression plasmids were introduced with the use of acationic lipid-based transfection reagent (293Fectin™; Invitrogen). Foreach transfection reaction, 20 μg of purified plasmids and 40 μL of the293Fectin™ were mixed with 1 mL of Opti-MEM® (Invitrogen) and incubatedfor 5 min at room temperature before being combined and allowed to formcomplexes for 20 min at room temperature. The DNA-293fectin complexeswere added to 3×10⁶ cells seeded in 90 mm petri plates and incubated at37° C., 8% CO2. In the final procedure, the supernatant was harvested 72hrs post-transfection by centrifugation (3,000 g, 15 min at 4° C.), torecover the secreted antibodies.

EXAMPLE 4 Epitope Mapping of the Invention Antibodies and AffinityDetermination

Using the technique described in Example 1 with respect to epitopemapping of the prior art antibody 131-2G, the epitopes corresponding tothe antibodies of the invention were determined. The affinity of theinvention antibodies was determined using the methods described inExample 1 with respect to mAb 131-2G.

As noted in Table 1 below, three of the antibodies bind a conformationalepitope—i.e., they do not map by binding overlapping peptides.Antibodies of the invention which map to specific sequences are shown inthe table. Also shown are the affinity constants, determined usingstandard Biacore assays with respect to recombinant Ga and Gb proteinsexpressed as pM, calculated from the measured on and off rates. The datafor two of these antibodies is shown in FIG. 6. Panels A, B and C showbinding data for 3G12 and panels D, E and F show the data from 3D3. Thetop row shows loading of the biosensor chip with antibody, the middlerow shows the signal arising from flowing Ga protein across the chipfollowed by washing with buffer, and the bottom row shows the same thingfor Gb protein. The increase in signal allows calculation of theon-rate, while the decrease during washing allows calculation of theoff-rate. The ratio of on to off rates is the affinity constant, Kd.

TABLE 1 KD × Ga KD × Gb Bin reactivity Epitope Epitope (pM) (pM) 1F12Ga/Gb 166-172 EVFNFVP 3G12 Ga/Gb 167-176 VFNFVPCSIC 579 173 1A5 Ga/Gb161-170 NDFHEEVFNF 3D3 Ga/Gb 164-172 HFEVFNFVP 1.1 3 1G1 Ga/Gbconformational 2B11 Ga/Gb 162-172 DFHFEVFNFVP 9 1.6 5D8 Ga/Gb 160-169NNDFHFEVFN 4390 1 2D10 Ga/Gb conformational 3F9 Ga Ga only 1D4 Ga/Gb165-171 FEVFNFV 230 52 1G8 Ga/Gb 161-170 NDFHFEVFNF 24 141 6A12 Ga/Gbconformational 10C6 Ga/Gb 164-168 *HFEVF 55 378 *same epitope as 131-2G

EXAMPLE 5 Comparison of Binding to G Protein with Binding to Virions

FIG. 7 shows the results of an ELISA assay employing live virus andassessing the binding using a standard horseradish peroxidase assay.Viral preps from various sources were used to coat plates at 10⁵PFU/well or higher concentration in carbonate buffer at pH 9.6 overnightat 4° C. Plates were blocked in 5% mik with PBST for an hour at roomtemperature. Serial dilutions of antibodies were added to wells inblocking buffer for one hour at room temperature. For detection, 1:2000dilution of goat anti-human Fc gamma—HRP (Jackson Immuno.) was added inblocking buffer for one hour at room temperature. Plates were washedextensively in PBST. Turnover of the substrate TMB was measured at 450nm. As shown in FIG. 7, 3D3 binds well to the live virus as do a numberof other antibodies of the invention. The Synagis® antibody, which hassubstantially weaker affinity, shows little binding to live virus evenat 10⁴ ng/ml antibody. FIG. 8 shows the correlation of the binding torecombinant protein as compared to binding to virus particles.

FIGS. 9A and 9B are graphs that demonstrate comparative ability of theantibodies of the invention to bind to strains A2 versus A5, using theassay described above. FIG. 9A shows that 3D3 and 3G12 bind well tostrain A2 as compared to Synagis®. PAB is a commercial polyclonal goatantibody against all RSV proteins (Chemicon, catalog#AB1128).

FIG. 9B shows that these antibodies also bind strain A5; note the unitson X-axis are different than in FIG. 9A. Similar experiments show thatthe antibodies of the invention bind to a wide variety of clinicalisolates.

EXAMPLE 6 Neutralization Assays

The ability of selected antibodies of the invention to neutralize virusin vitro was obtained by a standard plaque assay. HEp2 cells were platedin 12-well plates at 2×10⁵ cells/well. The following day, serialdilutions of antibodies were generated in media. Approximately 200PFU/well of RSV was added to the antibodies, in the presence of rabbitcomplement serum for one hour at room temperature. The antibody-virusmixture was then added to HEp2 cells at 200 uL/well for 2 hr at roomtemperature to allow for infection. Following this infection period,media were removed and media containing 1% methyl cellulose were addedto all wells. Plates were incubated at 35° C. for 6 days, after whichtime, cells were fixed and stained for plaque number determination, asfollows: Methyl cellulose is aspirated from the cell layers, and cellsare fixed in 100% methanol for 30 min at room temperature. The platesare then washed 3× with 5% milk in PBS. Primary antibody is added at1:500 dilution (Goat anti-RSV polyclonal antibody (Chemicon Cat#AB1128))in PBS+5% Milk Protein for 1 hr. Plates are washed again 3× with 5% milkin PBS. Secondary antibody is added at 1:500 dilution in 5% milk proteinin PBS (ImmunoPure Rabbit anti-goat antibody IgG (H+L) Peroxidaseconjugated) (Thermo Scientific, Cat#31402)) for 1 hr. Plates are washed3× with 1×PBS. Plaques are visualized by adding 1-Step Chloronaphtholsubstrate Pierce, Cat#34012), 200 μL per well for 10 min. Plates arerinsed with water and allowed to air dry. Plaques are counted in eachwell.

FIG. 10 shows the results in terms of absolute numbers of plaques per μgof human antibody and Synagis® antibody is included in the results.These data show that of the antibodies tested, 3D3 is most potent. 3G12has an IC₅₀ of 15 ng/ml or an affinity of 100 pM according to thisassay, whereas Synagis® commercial antibody has an IC₅₀ of 300 ng/mlcorresponding to an affinity of 2 nM. It was further found that Synagis®and the anti-G antibodies of the invention were not synergistic underthese conditions.

FIG. 11 shows neutralization of 3G12 antibody with respect to strain B,in comparison with Synagis®. The normalized data (% of control) arebased on an absolute plaque number of 160-180 per experiment. Theantibodies of the invention, with in vitro affinities from 1 pM up to 5nM (Table 1), have EC₅₀ values between 10-100 ng/ml.

EXAMPLE 7 Anti-G Prophylaxis in Mice

The invention antibodies, Synagis®, and human IgG1 were tested for theirability to prevent RSV infection in mice. On day −1, prior to infection,mice in the control group were injected i.p. with medium and PBS. Intest groups, injection was of 0.15, 1.5 and 15 mg/kg of antibodies hIgG1(non-immune, isotype control), or 3G12 or 3D3 or Synagis®. This amountsto approximately 3 μg, 30 μg and 300 μg per mouse.

On day 0, the mice were inoculated with 1×10⁶ pfu RSV long-strain byintranasal administration. On days 0 and 5, the lungs, bronchialalveolar lavage (BAL) and serum were collected and body weight, lungweight, pfu in the lung lobe section, viral load (by qPCR), lunghistology, total leukocytes, FACS, and IFNγ in BAL were all measured.

FIG. 12 shows the results based on viral lung load using the plaqueassay from the foregoing list. The data in FIG. 12 show that 3G12 and3D3 are equally effective as Synagis® in this assay. (A typical humandose for Synagis® is 15 mg/kg in humans.)

EXAMPLE 8 Therapeutic Efficacy of Antibodies to RSV-Ga/Gb

Antibodies to the conserved motif on RSV-G are shown to have therapeuticefficacy. Mice were infected intra-nasally at day 0 with 10⁶ pfu of RSV,then treated at day 3 with 3 mg/kg of antibody injected i.p. and assayedat days 5 and 7 for viral load in bronchial alveolar lavage. In thismodel, the infection is more readily cleared naturally than in humans.Nonetheless, the antibody treatment causes acceleration in viralclearance in a dose dependent fashion as compared to a control antibodythat does not bind RSV (FIG. 13A). Each treatment group had 5 animals,and the results are statistically significant.

As described in WO 00/43040, antibodies to Substance P are beneficial inalleviating the lung inflammation caused by RSV, an animal model for theprolonged morbidity that is the clinically important feature of RSVinfection. Up regulation of Substance P is dependent on active G protein(Haynes, L. M., et al., J. Virol. (2003) 77:9831-9844). Reduction inmeasures of lung inflammation following treatment with an antibody ofthe invention have also been observed, including reduction ininflammatory NK and total PMN cells (FIG. 13B), as well as reduction incytokines, e.g., IFNγ (FIG. 13C).

In an additional test, on day 0, mice were inoculated with 10⁶ pfu ofRSV A-type long strain by intranasal administration.

On day 3, various groups of 4-5 mice were treated as follows:

Group 1: control group which did not receive infection on day 0 and wastreated with PBS.

Group 2: negative control which received RSV inoculation on day 0 andPBS treatment on day 3.

Group 3: RSV inoculation on day 0 and Synagis® antibody i.p. in salineat 1, 10, or 100 μg per mouse or 0.05, 0.5 or 5 mg/kg.

Group 4: RSV inoculated on day 0 and administered mAb 3D3 in the sameprotocol as Group 3.

Group 5: received RSV inoculation on day 0 and administered 3G12 in thesame amounts as Groups 3 and 4.

Lungs and BAL fluid were collected on days 0, 3, 5, 7 and 10. Inaddition, body weight, lung weight, pfu in lung lobes, viral load byqPCR, lung histology, total leukocytes, FACS were measured as well asIFNγ in BAL. The results for qPCR in the groups administered 10 μg ofmAb are shown in FIG. 14.

As shown, the viral titer in Synagis®-treated and untreated mice behavessimilarly at this relatively low dose of antibody, whereas both 3D3treated and 3G12 treated mice had greatly lower titers at the peak ofinfection on day 5. This experiment verifies that higher affinity invitro correlates with higher potency in vivo.

FIG. 15 shows the dose response curve demonstrating that 3G12 and 3D3were able to lower the RSV copy number as measured by qPCR on day 7 atlower concentrations than Synagis®. 3D3 was particularly potent, againconsistent with having higher affinity in vitro.

Similarly, when qPCR viral counts are measured on day 10, although viraltiters are naturally very low at this point due to natural clearance bythe mouse immune system, 3D3 is approximately 100 times more potent thanSynagis® at the various dose concentrations as shown in FIG. 16. Thisexperiment highlights the utility of high affinity antibodies, whichcontinue to be effective even when the antigen concentration drops. Thehuman disease course is considerably more prolonged than in the mouse,providing a clear motivation for use of an antibody that continues toneutralize virus for an extended time period.

In still further experiments, mice were treated with murine anti-G mAbor murine anti-F mAb in groups of four, each experiment repeated threetimes. The mice were immunized on day 0 and treated with the antibodieson day 3, and various indications of efficacy were measured at days 3, 5and 7.

As one index of effectiveness, inflammatory cells in the bronchialalveolar lavage (BAL) were measured in the three groups with the resultsshown in FIG. 17. BAL cells per lung are plotted on the Y-axis from 0 to140×10³. The results show anti-F mAb lowered the BAL cells per lung atday 5 as compared to isotype control non-immune antibody, whereas anti-GmAb lowered the BAL cell count substantially more. By day 7, theinfection had run its course.

FIGS. 18A and 18B show a comparison of effectiveness of anti-G mAb(murine 131-2G) compared with anti-G F(ab′)₂ obtained from this antibodyby cleavage with pepsin and removal of the Fc fragments usingimmobilized Protein A. It has been shown that complement is importantfor the anti-viral effect of anti-G antibodies in vitro. This isconfirmed in FIG. 18A where the anti-viral effect is measured as pfu/glung tissue. Assays were conducted as in Example 6. The F(ab′)₂ fragmentof an anti-G antibody, which lacks the Fc portion of IgG that is neededfor complement mediated activity, is little better than control inlowering viral load, while anti-G mAb is very effective. However, wheninflammation is used as a measure of results, as shown in FIG. 18B, theF(ab′)₂ fragment of anti-G mAb is fully as effective as the completeantibody. This experiment establishes that neutralization of the Gprotein is critical to reducing airway inflammation. Since the virusactively secretes a soluble form of the G protein, and high affinitybinding is important for neutralization of soluble factors, the highaffinity antibodies of the invention are expected to have particularutility for the anti-inflammatory effect.

FIGS. 19A, B and C show the effect of anti-G mAb on the production ofIFNγ in BAL as a function of time of administration, with the cytokineserving as a marker for airway inflammation. Control non-immune antibodyin all cases fails to reduce the increase in IFNγ production thataccompanies airway inflammation. However, whether anti-G mAb isadministered at day −1 (panel A), at day +3 (panel B) or even at day +5(panel C), a dramatic decrease in the level of IFNγ at day 7 results.This experiment establishes utility of antibodies to the centralconserved motif of the RSV G protein for treating inflammation well pastthe peak of viral load.

EXAMPLE 9 Specificity of Endogenous Antibodies in Infected Subjects

Serum samples from four elderly adults with severe RSV disease and withsix elderly adults with mild RSV disease were tested forimmunoreactivity with the synthetic peptide:

which represents the conserved region of RSV G protein from strain A2.The assay was performed using the ELISA protocol described in Example 5.The levels of antibodies immunoreactive with this peptide correlate withthe severity of the disease wherein subjects with mild forms of thedisease exhibited much higher titers than subjects with more severemanifestations of the infection (see FIG. 20). These results indicatethat antibodies immunoreactive with this portion of the G protein areeffective in ameliorating infection.

1. An isolated monoclonal antibody (mAb) or immunoreactive fragmentthereof that: (a) binds an epitope within residues 160-176 of the Gprotein of respiratory syncytial virus (RSV) strain A2, (b) is minimallyimmunogenic when administered to human subjects, and (c) has reactivitywith both Ga and Gb, which mAb is, or which immunoreactive fragment isof, 3G12, 3D3, 2B11, 1D4, 1G8 or 10C6.
 2. The mAb of claim 1 which is acomplete antibody.
 3. A pharmaceutical composition that contains anisolated monoclonal antibody or fragment of claim 1, along with apharmaceutically acceptable excipient.
 4. A pharmaceutical compositionthat contains as sole active agents, the monoclonal antibody or fragmentof claim 1 and an additional pharmaceutical agent other than an antibodyimmunoreactive with RSV, along with a pharmaceutically acceptableexcipient.
 5. The composition of claim 4 wherein said additional agentis an anti-RSV drug.
 6. The pharmaceutical composition of claim 3 whichfurther contains one or more monoclonal antibodies or fragments thereofimmunoreactive with the F protein of RSV.
 7. The mAb of claim 1 that hasa therapeutic effect in a human subject infected with RSV.
 8. The mAb orfragment of claim 1 that reduces airway inflammation in a human subjectinfected with RSV.
 9. One or more nucleic acid molecules that comprisefirst nucleotide sequence(s) that encode(s) the mAb or fragment of claim1 or one or more nucleic acid molecule(s) that comprise(s) secondnucleotide sequence(s) complementary to said first nucleotidesequence(s) over its(their) entire length.
 10. Recombinant host cells orimmortalized cells that produce the mAb or fragment of claim
 1. 11. Therecombinant host cells of claim 10 which are mammalian cells, microbialcells, insect cells or plant cells.
 12. A method to produce an mAb orimmunoreactive fragment thereof, which method comprises culturing thecells of claim 10 and recovering said mAb or fragment.
 13. A transgenicnon-human animal or transgenic plant that produces the mAb or fragmentof claim
 1. 14. A method to treat RSV in a subject infected with RSV,which method comprises administering to a subject in need of suchtreatment and who is infected with RSV an effective amount of the mAb orfragment of claim
 7. 15. The method of claim 14 which results in reducedviral load.
 16. The method of claim 14 wherein the subject is human. 17.A method to treat RSV in a subject infected with RSV, which methodcomprises administering to a subject in need of such treatment and whois infected with RSV an effective amount of the pharmaceuticalcomposition of claim
 3. 18. The method of claim 17 which results inreduced viral load.
 19. The method of claim 17 wherein the subject ishuman.
 20. A method to enhance resistance to infection by RSV in a humansubject, which method comprises administering to a human subject in needof such resistance an effective amount of the mAb or fragment ofclaim
 1. 21. A method to enhance resistance to infection by RSV in ahuman subject, which method comprises administering to a human subjectin need of such resistance an effective amount of the pharmaceuticalcomposition of claim 3.